The detection of AChE inhibitors has been conducted using colorimetric methods that are based on decrease in color development by a primary or secondary substrate after exposure of the enzyme to a liquid sample containing an inhibitor. An example of such is the colorimetric method commercialized by Abraxis which uses Ellman's color reagent as an indicator substrate. That method tests the activity of a measured amount of enzyme after it has been exposed to a water sample potentially containing an inhibitor. After such exposure, the enzyme is reacted with its primary substrate, acetylthiocholine. Active enzyme will hydrolyze acetylthiocholine to thiocholine which in turn reacts with dithio-bis-nitrobenzoate to produce an orange-colored product, 5-thio-2-nitrobenzoate.
That method, though widely used, is complex, needing a standard curve for each test run, requiring numerous fluid transfer pipetting steps, and using a multiplicity of reaction vessels. In addition, the enzyme is inhibited by many organophosphates in vivo only after the inhibitor undergoes a physiological oxidation (Mahmoud and Casida, 1998). Hence, to mimic the required physiological oxidation for toxicity, a sample pretreatment step using an oxidizing agent, such as, bromine or hypochlorite, followed by a quencher to neutralize excess unreacted oxidizer, is required. Only by pre-oxidizing the sample can most organophosphates be detected by such methods.
Current laboratory procedures based on the use of Ellman's reagent that are approved include the Michel, microMichel, pH stat. Ellman and micro-Ellman assays, as well as certain variations of those assays.
Although those assays represent the current standard in AChE inhibitor detection. Ellman's reagent-based methods are not easily converted to rapid on-site testing because of the methodological complexity, the numerous steps and reaction vessels required and the need to keep heat-labile reagents refrigerated.
Other methods to detect such inhibitors are laboratory-based liquid, or gas chromatography methods coupled with mass spectrometry which rely on expensive, bulky equipment and elution profiles for identification.
Hence, there is a need for a simplified and rapid point of use assay. The instant invention addresses those shortcomings in the art. The test method of interest is simplified as a single device requiring only the addition of a measured volume of sample.